This study investigated the time course of changes in serum creatine kinase (CK), aspartate aminotransferase (AST), and alanine amino-transferase (ALT) activities after intramuscular injection of bupivacaine into the tibialis anterior (TA) of rats. Morphological changes in muscle cells, relationships between the amount of increase in the enzyme activities and the muscle mass damaged, and responses of serum enzymes to additional injections of bupivacaine hydrochloride (BPVC) were also examined. Adult male Wistar rats (24 wk) were placed into one of four groups. Group A (n = 7) was a control, and no injection was applied. Saline solution (0.5 ml of 0.9%) was injected into the right TA for group B (n = 5). BPVC (0.5 ml of 0.5%) was injected into the right TA for group C (n = 9) and into both the right and left TA for group D (n = 9). No increases in CK, AST, and ALT were observed for groups A and B. After BPVC injection, groups C and D showed significant (P < 0.01) increases in serum enzyme activities. CK peaked 4 h after BPVC injection, and AST and ALT peaked 12 h postinjection, then returned to the baseline by the time infiltration of mononuclear cells into the damaged muscle cells progressed. The amount of enzyme increase was significantly larger (P < 0.01) for group D compared with group C. Injection of BPVC into the right then into the left TA 4 h later displayed a bipolar response, and the second injection into the TA 12 wk after the first injection resulted in smaller increase in serum enzyme activities. It appeared that increases in serum enzyme activities reflected muscle damage; however, changes in enzymes occurred in the early stage of myonecrosis.